1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 JULY 2019 to 23 AUGUST 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The following deviation as already stated in study plan chapter 4.2 Selection of Animals: Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia, therefore no acclimation period of 48 h was performed. This is a deviation from OECD Guideline No. 202 and EU-Method C.2, which can be considered uncritical.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The following deviation as already stated in study plan chapter 4.2 Selection of Animals: Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia, therefore no acclimation period o

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: < 0.1 µg/L (see validation report VB18062903G926)
- Sampling method:
- Sample storage conditions before analysis:

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Eluate:
- Differential loading: <5%
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
- Other relevant information:

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Berlin
- Justification for species other than prescribed by test guideline:
- Age at study initiation (mean and range, SD): Between 0 and 24 hours
- Weight at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Stage and instar at study initiation:
- Valve height at study initiation, for shell deposition study (mean and range, SD):
- Peripheral shell growth removed prior to test initiation:
- Method of breeding: parthenogenesis
- Source: Umweltbundesamt Berlin
- Age of parental stock (mean and range, SD):
- Feeding during test:
- Food type: green algae (Desmodesmus subspticatus)
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration:
- Health/mortality:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20.4 – 21.8 °C

pH:

7.7

Dissolved oxygen:

The concentration of dissolved oxygen at the end of the test must be at least 3 mg/L.
The lowest concentration of dissolved oxygen at the end of the test was 8.3 mg/L.

Salinity:

Parameter /Concentration in mg/L
CaCl2*2H2O/ 293.80
MgSO4*7H2O/ 123.30
NaHCO3 /64.80
KCl /5.80

Nominal and measured concentrations:

0.1 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:glass beakers,
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: nominal volume 50 mL, tall shape, each containing 20 ± 5 mL test solution
- Volume of solution: 20 ± 5 mL test solution
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 5 Daphnia
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations: 0.1 µg/L (nominal concentration)
- Results used to determine the conditions for the definitive study: none Daphnia was immobilised

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (201902R201). The 24h-EC50 value was determined as 1.98 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L.

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight:
- Other biological observations:
- Mortality of control: none
- Other adverse effects control: none
- Immobilisation of control: no immobility of the daphnia has occurred
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium:

Results with reference substance (positive control):

The 24h-EC50 value was determined as 1.98 mg/L.
The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.

Reported statistics and error estimates:

Since no immobility of the daphnia has occurred, no statistical evaluation is carried out.

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed.
The solubility of the test item in test medium is < 0.1 µg/L (see validation report
VB18062903G926). Therefore, this study was performed as a limit test at 0.1 µg/L to show
that there is no toxicity at the LOQ (limit of quantification) of the analytical method. Acetone was used to spike the test solution. A stock solution containing 1 mg/L in acetone was
prepared. For preparing the treatment 100 µL/L stock solution was used.
For the solvent control, acetone in a concentration of 100 µL/L was used.
For the test concentration and the blank controls, 20 daphnia were exposed to the test
item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia
were counted.
None of the animals was immobilised in the blank and solvent controls and the treatment.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study to assure that the test conditions are reliable.
At the beginning and at the end of the test, the content of the test item in the test solutions
was determined using LC-MS-MS–determination. At the beginning of the test, the test item
could be detected very well. At the end of the test no test item could be detected. Very
probably it was taken up by the Daphnia. Therefore the results are related to the nominal
values.
Since no immobility of the daphnia has occurred, no statistical evaluation is carried out.
No observations were made which might cause doubts concerning the validity of the study
outcome. All validity criteria were met.
The result of the test is considered valid.

Description of key information

According to OECD 202 resp. EU C.2

The solubility of the test item in test medium is < 0.1 µg/L (see validation report
VB18062903G926). Therefore, this study was performed as a limit test at 0.1 µg/L to show that there is no toxicity at the LOQ (limit of quantification) of the analytical method. Acetone was used to spike the test solution. A stock solution containing 1 mg/L in acetone was prepared. For preparing the treatment 100 µL/L stock solution was used.
For the solvent control, acetone in a concentration of 100 µL/L was used.
For the test concentration and the blank controls, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.
The test item showed no toxicity. None of the animals was immobilised in the blank and solvent controls and the treatment.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.
At the beginning and at the end of the test, the content of the test item in the test solutions was determined using LC-MS-MS–determination. At the beginning of the test, the test item could be detected very well. At the end of the test no test item could be detected. Very probably it was taken up by the Daphnia. Therefore the results are related to the nominal values.
Since no immobility of the daphnia has occurred, no statistical evaluation is carried out.
The following results were determined for the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile (species: Daphnia magna).
48h-NOEC ≥ 0.1 µg/L (nominal)
48h-LOEC > 0.1 µg/L (nominal)
24h-EC50 > 0.1 µg/L (nominal)
48h-EC50 > 0.1 µg/L (nominal)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.1 µg/L